Dataset Information

Profiling gene expression levels to investigate the degree of substantial equivalence between GM and non-GM potato

Ontology highlight

ABSTRACT: The goal of this study was to calculate the degree of substantial equivalence between a GM potato line modified for late blight resistance and corresponding non-GM controls in the presence/absence of P. infestans inoculation. The GM phenotype was conferred through the introduction of the Rb gene which has been previously cloned from Solanum bulbocastanum (Song et al. 2003). The resulting GM phenotype was characterised by the expression of strong, partial resistance to P. infestans. By obtaining an expression profile from a GM and non-GM population before and after exposure to P. infestans, this effectively provides for the completion of two risk assessments: impact on the potato transcriptome of plus/minus genetic modification; before and after exposure to the pathogen. For each biological replicate, individuals from three separate GM lines (A, B, C) and three separate non-GM lines (D, E, F) were randomly sorted and cultivated for analysis. Each GM line contained an independent, single transgene insertion, which was verified through Southern Blot analysis. One day prior to pathogen exposure, the 4-week-old potato plants were transferred into a growth chamber with a 16-h light period, 90% relative humidity at a temperature of 15C. Plants were randomly selected and 14 individuals/line were inoculated with a field isolate of P. infestans. An additional 14 plants/line were sprayed with water. Inoculations were made with a sporangial suspension of 20,000/mL and plants were sprayed until run-off. Twenty-four hours post-inoculation, leaf tissue was collected from 9 plants per treatment, flash frozen in liquid nitrogen and pooled. Total leaf RNA was extracted using Trizol and the whole experiment was repeated three times (three biological replicates). Keywords: Direct comparison Overall design: 19 hybs total

Similar Datasets

Project description:The goal of this study was to calculate the degree of substantial equivalence between a GM potato line modified for late blight resistance and corresponding non-GM controls in the presence/absence of P. infestans inoculation. The GM phenotype was conferred through the introduction of the Rb gene which has been previously cloned from Solanum bulbocastanum (Song et al. 2003). The resulting GM phenotype was characterised by the expression of strong, partial resistance to P. infestans. By obtaining an expression profile from a GM and non-GM population before and after exposure to P. infestans, this effectively provides for the completion of two risk assessments: impact on the potato transcriptome of plus/minus genetic modification; before and after exposure to the pathogen. For each biological replicate, individuals from three separate GM lines (A, B, C) and three separate non-GM lines (D, E, F) were randomly sorted and cultivated for analysis. Each GM line contained an independent, single transgene insertion, which was verified through Southern Blot analysis. One day prior to pathogen exposure, the 4-week-old potato plants were transferred into a growth chamber with a 16-h light period, 90% relative humidity at a temperature of 15C. Plants were randomly selected and 14 individuals/line were inoculated with a field isolate of P. infestans. An additional 14 plants/line were sprayed with water. Inoculations were made with a sporangial suspension of 20,000/mL and plants were sprayed until run-off. Twenty-four hours post-inoculation, leaf tissue was collected from 9 plants per treatment, flash frozen in liquid nitrogen and pooled. Total leaf RNA was extracted using Trizol and the whole experiment was repeated three times (three biological replicates). Keywords: Direct comparison 19 hybs total

Project description:The late blight pathogen, Phytophthora infestans has a broad host range within the Solanaceae family, including yellow potato (Solanum phureja). The disease caused by P. infestans in S. phureja is poorly understood and is a major concern in Colombia. Expressed Sequence Tag (EST) libraries obtained from a normalized library constructed from healthy plant tissue revealed high levels of sequence similarity between S. phureja and S. tuberosum. Then, utilizing Serial Analysis of Gene Expression and high-throughput sequencing (SAGE-Solexa), we characterized yellow potato gene expression during infection by P. infestans. Four-week-old yellow potato plants were inoculated with P. infestans and were collected at 12 and 72 hours post inoculation for RNA extraction. We detected differentially expressed genes by comparing inoculated to non-inoculated and resistant to susceptible plants. The discovery and characterization of the proteins mediating this host–pathogen interaction enable the understanding of the pathosystem and is the key for developing resistant plants. Keywords: SAGE-Solexa, inoculation response, transcript profiling, Solanum phureja, Phytophthora infestans Overall design: Four-week-old yellow potato (Solanum phureja) plants were inoculated with Phytophthora infestans and were collected and flash frozen in liquid nitrogen at 12 and 72 hours post inoculation, as well as mock inoculated, for RNA extraction. 2 yellow potato cultivars (resistant and susceptible) were used for each experiment. Mock inoculated plants were collected in each replicate. RNA obtained from each of the three biological replicates was pooled to obtain a single RNA sample for each timepoint X cultivar combination. A total of 6 different SAGE libraries were thus obtained. For all libraries, Illumina sequencing was performed at Canada´s Michael Smith Genome Sciences Centre.

Project description:The late blight pathogen, Phytophthora infestans has a broad host range within the Solanaceae family, including yellow potato (Solanum phureja). The disease caused by P. infestans in S. phureja is poorly understood and is a major concern in Colombia. Expressed Sequence Tag (EST) libraries obtained from a normalized library constructed from healthy plant tissue revealed high levels of sequence similarity between S. phureja and S. tuberosum. Then, utilizing Serial Analysis of Gene Expression and high-throughput sequencing (SAGE-Solexa), we characterized yellow potato gene expression during infection by P. infestans. Four-week-old yellow potato plants were inoculated with P. infestans and were collected at 12 and 72 hours post inoculation for RNA extraction. We detected differentially expressed genes by comparing inoculated to non-inoculated and resistant to susceptible plants. The discovery and characterization of the proteins mediating this host–pathogen interaction enable the understanding of the pathosystem and is the key for developing resistant plants. Keywords: SAGE-Solexa, inoculation response, transcript profiling, Solanum phureja, Phytophthora infestans Four-week-old yellow potato (Solanum phureja) plants were inoculated with Phytophthora infestans and were collected and flash frozen in liquid nitrogen at 12 and 72 hours post inoculation, as well as mock inoculated, for RNA extraction. 2 yellow potato cultivars (resistant and susceptible) were used for each experiment. Mock inoculated plants were collected in each replicate. RNA obtained from each of the three biological replicates was pooled to obtain a single RNA sample for each timepoint X cultivar combination. A total of 6 different SAGE libraries were thus obtained. For all libraries, Illumina sequencing was performed at Canada´s Michael Smith Genome Sciences Centre.

Project description:Late blight, caused by the oomycete Phytophthora infestans, is one of the most damaging potato diseases. Genetic resistance is one of the most effective means to control the destruction caused by this pathogen. Transgenic potato lines harboring a resistance gene, RB, confer broad-spectrum, rate-reducing late blight resistance. A microarray approach was used to understand what genes are manipulated in the potato background after the addition of the RB gene that contribute to the late blight resistant phenotype. Keywords: Time course, disease state analysis Overall design: CRD (3x2x2) Split-Split Plot: 3 sampling time points after inoculation (2, 5, 10 hours), Two genotypes (Katahdin with and without the RB gene), Inoculation with P. infestans or mock inoculation with water. 48 arrays were hybridized in total; 12 in each biological replicate. Each genotype with the mock and late blight inoculated samples was hybridized on two arrays using a dye-swap procedure. Each genotype had a total of 6 arrays across the three sampling time points.

Project description:The expression of genes in P. infestans was monitored over a five day time course of a potato infection. Genes encoding known and putative effector proteins were found induced at two days post-inoculation with expression decaying over the five day time course. Overall design: The expression of genes in P. infestans was monitored over a five day time course of a potato infection. Genes induced or repressed during infection were identified by comparing expression intensities derived from samples of infected potato to baseline expression intensities provided by samples derived from mycelia growing in media (Pea, V8, or RS agar). Biological replicates were generated for each sample. Total RNA samples were submitted to NimbleGen for full microarray expression services, including cDNA conversion, Cy3 labeling, hybridizations, and scans.

Project description:Late blight, caused by the oomycete Phytophthora infestans, is one of the most damaging potato diseases. Genetic resistance is one of the most effective means to control the destruction caused by this pathogen. Transgenic potato lines harboring a resistance gene, RB, confer broad-spectrum, rate-reducing late blight resistance. A microarray approach was used to understand what genes are manipulated in the potato background after the addition of the RB gene that contribute to the late blight resistant phenotype. Keywords: Time course, disease state analysis CRD (3x2x2) Split-Split Plot: 3 sampling time points after inoculation (2, 5, 10 hours), Two genotypes (Katahdin with and without the RB gene), Inoculation with P. infestans or mock inoculation with water. 48 arrays were hybridized in total; 12 in each biological replicate. Each genotype with the mock and late blight inoculated samples was hybridized on two arrays using a dye-swap procedure. Each genotype had a total of 6 arrays across the three sampling time points.

Project description:The expression of genes in P. infestans isolates 06_3928A and NL07434 was monitored from 2 to 4 days time course of a potato infection. Genes encoding known and putative effector proteins were found induced at two and/or three days post-inoculation. We used a custom chip GPL8093 from Roche Nimblegen’s proprietary Maskless Array Synthesis (MAS) technology uses digital light processing and rapid, high-yield photochemistry to synthesize long oligo, high-density DNA microarrays with extreme flexibility. Each GPL8093 chip measures the expression level of 18155 genes from Phytophthora infestans with 60-mer probe pairs (PM/MM) per gene. Total RNA samples recovered from mycelia in two medias (rye sucrose agar and V8 agar) and infected potato leaves from a time couse infection (2, 3 and 4 days post incoulation). Experiments included two biological repllicates from each sample. We carried out total RNA extractions (mycelia and potato infected material) for two P. infestans isolates 06_3928A and NL07434. cDNA synthesis was performed Nimblegen.

Project description:The oomycete P. infestans is the causal agent of late blight, the most devastating potato disease. In contrast to potato, A. thaliana is able to successfully prevent colonization of the pathogen due to a multi-layered nonhost resistance. Several mutants have been isolated which are impaired in penetration resistance. A mutation in the gene PEN2, which encodes for an enzyme involved in indole glucosinolate metabolism (Bednarek et al. (2009)), results in the loss of penetration resistance against P. infestans (Lipka et al. (2005)). Despite its ability to penetrate epidermal cells of pen2 mutant plants, P. infestans is still not able to colonize these plants. Additional mutants were isolated by Kopischke et al. (2013) which show enhanced defense responses upon infection with P. infestans: pen2erp1 and pen2erp2, and backcrossed mutants erp140 and erp2D. We used six different plant lines, the wildtype-like gl1, and the five different mutants (pen2, pen2erp1, pen2erp2, erp2D, erp140). The plants were either infected with P. infestans spores or treated with water as control, and harvested 6h and 12h after treatment. The experiment was repeated three times with different P. infestans cultures, resulting in biological triplicates, for an overall of 6 x 2 x 2 x 3 = 72 samples. A metabolomics data set from the same set of samples has been submitted to MetaboLights database at EMBL-EBI (http://www.ebi.ac.uk/metabolights/index) under accession number MTBLS18 .

Project description:Infection by Albugo species, resulting in white rust disease, suppresses host plant immunity, and can even enable Albugo laibachii-infected Arabidopsis to support growth and reproduction of the non-host potato late blight pathogen Phytophthora infestans. However, the mechanisms involved in non-host resistance remain to be elucidated. Here, we investigate specific host defense mechanisms that are suppressed by A. laibachii and A. candida infection, and compare the resistance contributed by indole glucosinolates and camalexin to that resulting from other components of defense induced by salicylic acid. We conclude a broad repertoire of host defense components contributes to non-host resistance in Arabidopsis to P. infestans, with a particular role for tryptophan-derived anti-microbial metabolites. Identifying the mechanisms involved in non-host resistance to pathogens such as P. infestans is necessary in the development of strategies to ensure future food security. Overall design: 4-5 weeks old Arabidopsis (MAGIC-107: a multi-parent recombinant inbred line) plants were spray inoculated with either Mock or Albugo candida strain Nc2 or Albugo laibachii strain Nc14 , and infected plants were harvested at 0, 2, 4, 6 and 8 days post-inoculation (dpi) for total RNA extraction. mRNA profiles were generated by deep sequencing on Illumina GAIIx using EXPRSS tag-seq protocol. Four biological replicates were collected. Samples of Nc2 and Nc14 infections from 2, 4, 6 and 8 dpi were sequenced each in a separate lane. Samples of 2 and 4 dpi are sequenced 3 times and 6 dpi samples are 2 times to generate higher depth of sequencing data. Samples of Mock, Nc2 and Nc14 infections from 0 dpi were sequenced in one lane. Mock samples from 2, 4, 6 and 8 dpi were sequenced in one lane.